Sequence-specific binding of oligonucleotides both to single-stranded and to duplex DNA has been recognized. The appropriate sequence recognition for binding to single-stranded targets is well known: the A-T and G-C pairing characteristic of duplex formation has been established as the basis for DNA replication and transcription. More recently, it has been realized that oligonucleotides may also bind in a sequence-specific manner to duplex DNA in order to form triplexes.
Thus, duplex DNA can be specifically recognized by oligomers based on a recognizable nucleotide sequence. Two major recognition motifs have been recognized. In an earlier description of a "CT" motif, cytosine residues recognize G-C basepairs while thymine residues recognize A-T basepairs in the duplex. These recognition rules are outlined by Maher III, L. J., et al., Science (1989) 245:725-730; Moser, H. E., et al., Science (1987) 238:645-650. More recently, an additional motif, called "GT" recognition, was described by Cooney, M., et al., Science (1988) 241:456-459; Hogan, M. E., et al., EP Publication 375408. In the G-T motif, A-T pairs are recognized by adenine or thymine residues and G-C pairs by guanine residues.
In both of these binding motifs, the recognition sequence must align with a sequence as played out on one of the chains of the duplex; thus, recognition, for example, of an A-T pair by a thymine depends on the location of repeated adenyl residues along one chain of the duplex and thymine series on the other. The recognition does not extend to alternating A-T-A-T sequences; only the adenyl residues on one chain or the other would be recognized. An exception to the foregoing is the recent report by Griffin, L. C., et al., Science (1989) 245:967-971, that limited numbers of guanine residues can be provided within pyrimidine-rich oligomers and specifically recognize thymine-adenine base pairs; this permits the inclusion of at least a limited number of pyrimidine residues in the homopurine target.
The two motifs exhibit opposite binding orientations with regard to homopurine target chains in the duplex. In the CT motif, the targeting oligonucleotide is oriented parallel to the target purine-rich sequence; in the GT motif, it is oriented antiparallel (Beal, P. A., et al., Science (1990) 251:1360-1363). Thus, recognition sequences in the CT motif are read with respect to target 5'.fwdarw.3' sequences so that in the 5'.fwdarw.3' direction, synthetic oligonucleotides contain the required sequence of C or T residues with respect to the guanine or adenyl residues in the target. In the GT motif, on the other hand, the targeted sequence is read 5'.fwdarw.3' in order to design the 3'.fwdarw.5' sequence of the targeting oligonucleotide.
One problem that has arisen with respect to binding in the CT system resides in the ionization state of the "C" residue at neutral or physiological pH. In order to form the appropriate hydrogen bond donor/acceptor pattern, the amino group at position 3 of the C must be protonated. This is consonant with the pK.sub.a when the pH is low (cytosine pK.sub.a is 4.25), but at neutral pH, most of the pyrimidines are unprotonated. This interferes with binding at physiological pH.
One proposed solution to this problem has been the use of 5-methylcytosine (pK.sub.a 4.35) instead of cytosine as the recognizing "C". This approach was based upon the observation (Lee, J. S. et al., Nucleic Acids Res (1984) 12:6603-6614) that polypyrimidine oligonucleotides composed of 5-methyldeoxycytidine can bind to poly G:poly C double-stranded DNA at neutral pH. The ability of both 5-bromouracil and 5-methylcytosine to bind duplex DNA at the same homopurine target sequence as their T/C analogs, but with greater affinities and over an extended pH range has also been reported by Povsic, T. J., et al., J Am Chem Soc (1989) 111:3059-3061. The improved binding of 5-methylcytosine compared to cytosine in CT mode binding is believed to result from (i) an increased pK.sub.a, and (ii) interaction of the methyl group at position 5 with adjacent methyl groups in the oligomer. Another approach which was taken (Cooney, M.; Czernuszewicz, G.; Postel, E. H.; Flint, E. S. J.; and Hogan, M. E. Science (1988) 241:456-459) was the substitution of deoxyguanosine for deoxycytidine, and the substitution of deoxyadenosine for thymidine to yield an alternative binding motif.
Sequence-specific targeting of both single-stranded and duplex oligonucleotides has applications in diagnosis and analysis. Under some circumstances wherein such binding is to be effected, it is advantageous to stabilize the resulting duplex or triplex over long time periods.
Covalent crosslinking of the oligomer to the target provides one answer to this problem. Sequence-specific recognition of single-stranded DNA accompanied by covalent crosslinking has been reported by several groups. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986) 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. A report of similar work by the same group is that by Knorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, R. B., et al., J Am Chem Soc (1989) 111:8517-8519, effect covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. A photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Home, et al., J Am Chem Soc (1990) 112:2435-2437.
Use of N.sup.4, N.sup.4 -ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides has also been described by Webb and Matteucci, J Am Chem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674. These papers also describe the synthesis of oligonucleotides containing the derivatized cytosine. Matteucci and Webb, in a later article in Tetrahedron Letters (1987) 28:2469-2472, describe the synthesis of oligomers containing N.sup.6, N.sup.6 -ethanoadenine and the crosslinking properties of this residue in the context of an oligonucleotide binding to a single-stranded DNA.
In a recent paper, Praseuth, D., et al., Proc Natl Acad Sci (U.S.A.) (1988) 85:1349-1353, described sequence-specific binding of an octathymidylate conjugated to a photoactivatable crosslinking agent to both single-stranded and double-stranded DNA. A target 27-mer duplex containing a polyA tract showed binding of the octathymidylate in parallel along the polyA. Photoactivated crosslinking of the duplex with a p-azidophenacyl residue covalently linked to the terminus of the octathymidylate was achieved. While sequence-specific association occurred at the predicted region of the duplex, it appeared that the crosslinking reaction itself was not target specific. As photoactivation was required to form the covalent crosslink, there could be no question of accurate sequence-specific association of the octathymidylate to the target sequence in the 27-mer duplex. A requirement for photoactivation, however, seriously limits the therapeutic potential of the crosslinking agent. Administration to a live subject does not readily admit of this mechanism of action.
In addition, Vlassov, V. V. et al., Gene (1988) 313-322 and Fedorova, O. S. et al., FEBS (1988) 228:273-276, describe targeting duplex DNA with a 5'-phospho-linked oligonucleotide.
In effecting binding to obtain a triplex, to provide for instances wherein purine residues are concentrated on one chain of the target and then on the opposite chain, oligonucleotides of inverted polarity may be provided. By "inverted polarity" is meant that the oligonucleotide contains tandem sequences which have opposite polarity, i.e., one having polarity 5'.fwdarw.3' followed by another with polarity 3'.fwdarw.5', or vice versa. This implies that these sequences are joined by linkages which can be thought of as effectively a 3'--3' internucleotide junction (however the linkage is accomplished), or effectively a 5'--5' internucleotide junction. Such oligomers have been suggested as by-products of reactions to obtain cyclic oligonucleotides by Capobionco, M. L., et al., Nucleic Acids Res (1990) 18:2661-2669. Compositions of "parallel-stranded DNA" designed to form hairpins secured with AT linkages using either a 3'--3' inversion or a 5'--5' inversion have been synthesized by van de Sande, J. H., et al., Science (1988) 241:551-557. In addition, triple helix formation using an oligomer which contains an effective 3'--3' linkage has been described by Horne, D. A., and Dervan, P. B., J Am Chem Soc (1900) 112:2435-2437.
Single-stranded nucleic acid, primarily RNA, is the target molecule for oligonucleotides that are used to inhibit gene expression by an "antisense" mechanism (Uhlmann, E., et al, Chem Reviews (1990) 90:543-584; van der Krol, A. R., et al, Biotechniques (1988) 6:958-976). Antisense oligonucleotides are postulated to exert an effect on target gene expression by hybridizing with a complementary RNA sequence. The hybrid RNA-oligonucleotide duplex appears to interfere with one or more aspects of RNA metabolism including processing, translation and metabolic turnover. Chemically modified oligonucleotides have been used to enhance their nuclease stability.